In the present grant period we have made excellent progress in three general areas: understanding the mechanism of inhibition of HIV-induced membrane fusion by class A amphipathic helixes, developing a theory to explain the mechanism of action of the HIV fusion peptide, and understanding the properties of the amphipathic helixes in gp41. Specifically, we have made the following findings: (a) Class A amphipathic helixes are powerful inhibitors of membrane fusion induced by HIV and other enveloped viruses. (b) Class A amphipathic helixes stabilize bilayers, inhibit fusion of model membranes and inhibit membrane lysis by class L (lytic) amphipathic helixes; the latter appear to destabilize the bilayer lipid phase by stabilizing inverted lipid phases. (c) A molecular model (the reciprocal wedge hypothesis) that effectively explains the reciprocal effects of class A and class L amphipathic helixes on membranes was developed and tested. (d) A novel molecular model for the mechanism of action of HIV fusion peptides, called the beta-wedge, has been proposed that is inspired by the reciprocal wedge hypothesis; preliminary CD studies indicate that peptide analogs of viral fusion peptides have beta structure in solution. (e) Peptide analogs of the two C-terminal amphipathic helixes of gp41 were found to inhibit HIV-induced membrane fusion and were cytotoxic to cells. (f) Peptide analogs of the two gp41 amphipathic helixes (and gp160 but not gp120) were shown to bind to calmodulin and to inhibit its activity in vitro. (g) HDL was found to be significantly lower in asymptomatic HIV positive patients than in controls but the clinical significance of this observation has not been established. We propose the following specific aims: (1) Elucidate the basic molecular mechanism of action of class A and class L amphipathic helixes to produce peptide and nonpeptide analogs with increasing abilities to stabilize or destabilize bilayers. (2) Investigate the molecular mechanism of action of the HIV fusion peptide to develop new anti-fusion peptides and nonpeptide antagonists. (3) Continue our studies of the properties and function of amphipathic helixes in gp4l. New methodological directions include: (a) collaborations with Drs. Bill Gallaher and Eric Hunter to explore molecular mechanisms of fusion and fusion inhibition through the use of site-directed mutagenesis, and (b) Silicon Graphics-based molecular modeling of fusion inducing and inhibiting peptides and non-peptide antagonists.